p-Toluenesulfonyl Chloride Catalysed Facile Synthesis of O-benzyl-l-amino Acids and Their In Vitro Evaluation

Article abstract of DOI:10.1007/s10989-019-09998-x

Protection and subsequent deprotection of amino acid functional groups play a key role in regioselective peptide synthesis. For protection, carboxylic acid functional groups are often benzylated using p-toluenesulfonic acid catalysed Fischer-Speier esterification reaction. Such reaction involves in situ water formation, which requires subsequent separation by azeotropic distillation for forward shift of equilibrium. To eliminate the need of this corresponding step requiring additional set-up, current study investigated p-toluenesulfonyl chloride as a reasonable alternative catalyst for facile benzylation of selected mono- and di- carboxylic amino acids. Literature reports that p-toluenesulfonyl chloride not only has a better shelf life but also demonstrates better safety in case of accidental systemic absorption over p-toluenesulfonic acid. As the O-benzyl-l-amino acids are often retained without deprotection to constitute the pharmaceutical peptide systems, synthesized compounds were investigated for their biocompatibility using in vitro cytotoxicity assays.

Full text of DOI:10.1007/s10989-019-09998-x

International Journal of Peptide Research and Therapeutics
https://doi.org/10.1007/s10989-019-09998-x
p‑Toluenesulfonyl Chloride Catalysed Facile Synthesis
of O‑benzyl‑l‑amino Acids and Their In Vitro Evaluation
Namita Hegde1 · Kapil Juvale1 · Bala Prabhakar1
Accepted: 18 December 2019
© Springer Nature B.V. 2020
Abstract
Protection and subsequent deprotection of amino acid functional groups play a key role in regioselective peptide synthesis.
For protection, carboxylic acid functional groups are often benzylated using p-toluenesulfonic acid catalysed Fischer-Speier
esterifcation reaction. Such reaction involves in situ water formation, which requires subsequent separation by azeotropic
distillation for forward shift of equilibrium. To eliminate the need of this corresponding step requiring additional set-up,
current study investigated p-toluenesulfonyl chloride as a reasonable alternative catalyst for facile benzylation of selected
mono- and di- carboxylic amino acids. Literature reports that p-toluenesulfonyl chloride not only has a better shelf life but also
demonstrates better safety in case of accidental systemic absorption over p-toluenesulfonic acid. As the O-benzyl-l-amino
acids are often retained without deprotection to constitute the pharmaceutical peptide systems, synthesized compounds were
investigated for their biocompatibility using in vitro cytotoxicity assays.
Keywords Amino acids · Benzylation · Esterifcation · Peptide synthesis
Introduction
its dissociation from synthesized moiety i.e., deprotection
should be easy and harmless (Isidro-Llobet et al. 2009).
Peptides often constitute the pharmaceutical delivery sys-
tems. Protection and subsequent deprotection of amino acid
functional groups play a key role in peptide synthesis. As
each amino acid individually contain one or more carboxylic
acid and amine functional groups, their planned protection
becomes necessary for regioselective peptide synthesis. Car-
boxylic acid functional groups are protected through their
esterifcation, owing to simplicity of the reaction, product
stability as well as inertness during coupling reactions and
ease of deprotection at fnal stage to obtain the desired com-
pound. Hence, amino acid esters form important intermedi-
ates for peptide synthesis.
Organic synthesis requires permutation of reagents and cata-
lysts for clean synthesis of desired products, without forma-
tion of undesired products through side reactions. Protecting
groups are often used to diminish such side reactions; since
these are the molecules that mask and exclude extra func-
tional groups from reacting during synthesis. This is one of
the approaches undertaken to achieve regioselective bond
formation in organic synthesis. A chemical with the follow-
ing three fundamental properties can be used as a protecting
group in organic synthesis: (i) its association with target
functional group should be easy, (ii) the association should
remain stable over a broad range of reaction conditions, (iii)
Fischer-Speier esterifcation reaction is often employed
for protecting the carboxylic acid functional group. The
reaction (Fig. 1) is used for derivatizing carboxylic acid
function to its methyl, ethyl, benzyl etc. esters by refuxing
the former with excess of respective alcohol in presence of
an acidic catalyst such as hydrochloric acid (HCl), sulfuric
acid (H2SO4) or p-toluenesulfonic acid (p-TsOH). Due to
the better purity of esters formed and ease of catalyst neu-
tralisation during product purifcation, p-TsOH is preferred
over HCl and H2SO4 (Furniss et al. 1996). As esterifca-
tion employing alcohol and carboxylic acid is reversible,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10989-019-09998-x) contains
supplementary material, which is available to authorized users.
* Kapil Juvale
kjuvale@gmail.com
* Bala Prabhakar
bala.prabhakar@nmims.edu
1
Shobhaben Pratapbhai Patel School of Pharmacy &
Technology Management, SVKM’s NMIMS, V. L. Mehta
Road, Vile Parle (W), Mumbai 400056, India
Vol.:(0123456789)
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International Journal of Peptide Research and Therapeutics
both forms assume deliquescent nature on long atmos-
pheric exposures, as compared to the anhydrous form
(m.p. 38 °C) its monohydrate form (m.p. 103–106 °C)
is more stable. However, due to its hygroscopic nature
the monohydrate compound is often observed to absorb
moisture during storage and turn pink in colour. Hence,
shelf life of p-TsOH is highly infuenced and limited by
the storage conditions practiced (SDS 2018a). Alterna-
tively, due to its water insolubility and anhydrous nature,
p-TsCl demonstrates better stability and subsequently bet-
ter shelf life over p-TsOH (SDS 2018b). Moreover, based
on results of toxicity studies conducted in the rodent mod-
els, literature indicate that in case of accidental systemic
absorption p-toluenesulfonyl chloride (p-TsCl) (oral-rabbit
LD50: 4680 mg/kg) is safer than p-TsOH (oral-rat LD50:
2480 mg/kg) (Chemicalbook 2019a, b).
O
O
H+
OR2
H2O
+
R1
R2 OH
+
OH
R1
Fig. 1 Fischer-Speier esterifcation reaction
an equilibrium exists; which can be rapidly driven forward
towards ester formation by azeotropic distillation of water
formed in situ. Hence, Fischer–Speier esterifcation reac-
tion is often carried out in presence of organic solvents like
benzene or toluene forming heterogeneous azeotrope with
water (Furniss et al. 1996; Wang 2010).
Bolchi et al. investigated the impact of water azeotroping
solvents employed for Fischer-Speier esterifcation reaction
on environmental safety and product racemization. The sig-
nifcant observations reported were (i) water azeotroping
solvents such as benzene (b.p. 80.1 °C) and carbon tetra-
chloride (b.p. 76.72 °C) are environmentally hazardous, and
(ii) high-boiling, water azeotroping solvents such as toluene
(b.p. 110.6 °C) or neat benzyl alcohol (b.p. 205 °C) give
scope for product racemization. Although cyclohexane (b.p.
81 °C) was reported as a suitable solvent, azeotropic distil-
lation of water was still necessary as corresponding step
requiring additional set-up (Bolchi et al. 2015, 2017, 2018).
p-TsOH is a white, crystalline, water soluble solid avail-
able in its anhydrous and monohydrate forms. Although
Hence current study (Fig. 2) investigated application of
p-TsCl as an alternative catalyst for benzylation of selected
mono- and di- carboxylic amino acids. Use of p-TsCl not
only eliminated the need of tedious azeotropic distilla-
tion, but also presented an alternative catalyst over p-TsOH
with better shelf life and safety (in case of accidental sys-
temic absorption). Furthermore, biocompatibility of the
formed products was investigated using in vitro cytotoxic-
ity assays; since O-benzyl-l-amino acids are often retained
without deprotection to constitute the pharmaceutical pep-
tide systems.
A) Previous Work: (1) Bolchi's Work (Bolchi et al. 2015) and (2) Arai's Work (Arai and Muramatsu 1982)
O
OH
S
O
O
O
(1)
O
O
p-TsOH, cyclohexane
Reflux with Dean-Stark trap
2
O
O
n
HO
OH
n
OH
NH2
NH2
Fischer-Speier esterification
H
N
(2)
H
N
p-TsCl, p-TsOH
OH
80oC
NH2 HCl
NH2
OH
OH
O
O
Transesterification
B) Present Work:
O
S
OH
O
O
O
(3)
O
O
p-TsCl, cyclohexane
O
O
n
2
HO
OH
n
OH
NH2
Reflux
NH2
Transesterification and Fischer-Speier esterification
Fig. 2 Summary of diference between previous work and present work
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International Journal of Peptide Research and Therapeutics
Experimental
mixture contained 1 gm (6.8 mmol) l-glutamic acid, 1.56
gm (8.2 mmol) p-TsCl, 4 ml (38.6 mmol) benzyl alcohol
and 20 ml (excess) cyclohexane. The reaction mixture was
refuxed overnight under continuous stirring and progress
of reaction was monitored through TLC. On completion,
the reaction mixture was cooled to room temperature and
ethyl acetate was added till complete precipitation of prod-
ucts 1a–1c. The precipitate obtained was fltered, washed
with cold ethyl acetate and dried to yield benzyl-protected
p-toluenesulfonate salts of respective amino acids.
Materials and Methods
Chemistry
Extra pure grade l-glutamic acid, l-aspartic acid and l-phe-
nylalanine were purchased from S. D. Fine Chemical Lim-
ited (India). The analytical grade solvents and p-TsCl were
procured from Research-Lab Fine Chem Industries (India).
Reagent grade benzyl alcohol and cyclohexane were ordered
from Thermo Fisher Scientifc India Pvt. Ltd. (India) and
Spectrochem Pvt. Ltd. (India), respectively. Progress of
the reaction was monitored by thin-layer chromatography
(TLC) using silica gel 60 F254 supported on aluminium plate
(Merck). Fourier-Transform infrared (FTIR) spectra of the
synthesized molecules were recorded using Perkin Elmer
Spectra RXI FTIR spectrometer. Mass spectra of synthe-
sized molecules were recorded using Shimadzu LCMS-8040
triple quadrupole LC–MS/MS spectrometer having Electro-
spray Ionisation (ESI) source. Nuclear Magnetic Resonance
(NMR) spectra of synthesized molecules were recorded
using Bruker NMR spectrometer. Mettler Toledo DSC 1
STARe Thermogravimetric Analyser was used to perform
thermal analysis of all the synthesized compounds.
l-Glutamic acid dibenzyl ester p-toluenesulfonate (1a):
obtained as a white solid (2.48 g, 81%) m.p. 130.31 °C, Rf
(TLC, methanol/ dichloromethane 1:9) = 0.83, IR (KBr,
cm−1): 3471.40 (N–H stretching), 1743.30, 1731.58 (C=O
1
stretching), 1599.39 (N–H bending). H NMR (500 MHz,
DMSO-d6) δ 8.41 (s, 3H), 7.51 (d, J = 8.1 Hz, 2H),
7.48–7.33 (m, 10H), 7.13 (d, J=7.6 Hz, 2H), 5.32–5.18 (m,
2H), 5.11 (s, 2H), 4.18 (t, J=6.5 Hz, 1H), 2.61 (q, J=8.4,
7.7 Hz, 2H), 2.30 (s, 3H), 2.12 (tq, J=14.6, 8.1, 7.3 Hz, 2H).
MS (ESI) m/z calcd for C19H22NO4+ [M+H] + 328.15; found
328.25; MS (ESI) m/z calcd for C7H7SO3− [M–H]− 171.01,
found 170.40.
l-Aspartic acid dibenzyl ester p-toluenesulfonate (1b):
obtained as a white solid (3.17 g, 87%) m.p. 159.95 °C, Rf
(TLC, methanol/ dichloromethane 1:9) = 0.87, IR (KBr,
cm−1): 3455.46 (N–H stretching), 1758.20, 1734.26 (C=O
1
stretching), 1593.17 (N–H bending). H NMR (400 MHz,
Synthesis of 1a–1c
DMSO-d6) δ 8.45 (s, 2H), 7.46 (d, J = 8.1 Hz, 2H),
7.40–7.27 (m, J=3.9, 3.3 Hz, 10H), 7.08 (d, J=7.8 Hz, 2H),
5.16 (s, 2H), 5.07 (s, 2H), 4.46 (t, J=5.4 Hz, 1H), 2.99 (qd,
J=17.5, 5.4 Hz, 2H), 2.25 (s, 3H). MS (ESI) m/z calcd for
C18H20NO4+ [M+H]+ 314.14, found 314.10; MS (ESI) m/z
calcd for C7H7SO3− [M–H]− 171.01, found 170.90.
General procedure for synthesis of 1a–1c: p-TsCl catalysed
benzyl esterifcation of mono- and di- carboxylic acid is
described in Scheme 1. For l-phenylalanine, since it con-
tains monocarboxylic group, half equivalents of benzyl alco-
hol and p-TsCl were taken than those used for dicarboxylic
amino acids l-aspartic acid and l-glutamic acid. Reaction
l-Phenylalanine benzyl ester p-toluenesulfonate (1c):
obtained as a white solid (1.89 g, 73%) m.p. 164.91 °C, Rf
O
S
OH
(A)
O
O
O
O
O
p-TsCl, cyclohexane, 24 hr
O
O
n
2
HO
OH
n
OH
NH2
Reflux
NH2
1a
1b
, n= 1
, n= 0
O
S
(B)
OH
O
O
O
p-TsCl, cyclohexane, 24 hr
O
OH
OH
NH2
NH2
Reflux
1c
Scheme 1 Synthesis of O-benzyl amino acids, a dibenzylation of l-glutamic acid (1a) and l-aspartic acid (1b) and b benzylation of l-phenylala-
nine (1c)
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International Journal of Peptide Research and Therapeutics
(TLC, methanol/dichloromethane 1:9) = 0.285, IR (KBr,
cm−1): 3450.98 (N–H stretching), 1741.29 (C =O stretch-
ing), 1602.79 (N–H bending). 1H NMR (400 MHz, DMSO-
d6) δ 8.44 (s, 3H), 7.72–6.78 (m, 14H), 5.18–5.05 (m, 2H),
4.35 (t, J = 6.1 Hz, 1H), 3.22–2.95 (m, 2H), 2.26 (s, 3H).
MS (ESI) m/z calcd for C16H18NO2+ [M+H]+ 256.13, found
256.10; MS (ESI) m/z calcd for C7H7SO3− [M–H]− 171.01,
found 171.00.
Results and Discussion
Synthesis
In the current study p-TsCl was used to synthesize p-toluene-
sulfonates of selected mono- and di- carboxylic amino acid
esters without employing azeotropic distillation of water.
In the synthesis, benzyl alcohol was used as a benzylating
agent, cyclohexane was used as a low-boiling solvent and
p-TsCl was used as a catalyst for transesterifcation. Since
similar reaction using p-TsOH catalyst was also reported
by Bolchi et al. (Bolchi et al. 2015), we compared practical
yields of L-glutamic acid esterifcation obtained by both the
methods, keeping equivalent ratios of all the components
same. % yield of former was 86.69% whereas that of latter
was 80.80%, indicating acceptable yields with p-TsCl with-
out employing azeotropic distillation.
Cytotoxicity Study
Intrinsic cytotoxicity of the synthesized compounds was
determined by MTT assay using A549 (adenocarcinoma)
and T3T (fbroblast) cells. MTT assay was performed as
described earlier with minor modifcations (Juvale et al.
2013). Briefy, cells were trypsinised at 90% confuence
and 5000 cells/well were seeded into 96-well plates. The
plates were then incubated for 24 h at 37 °C and 5% CO2.
After the incubation, test compounds were added at fnal
concentration of 25 µM in triplicates and the plates were
further incubated for 72 h. MTT dye (5 mg/ml) was then
added to each well and incubated for next 2 h. The superna-
tant was removed and 100 µL of DMSO was added to each
well to dissolve formazan crystals formed. Optical density of
formazan was measured at 570 nm using Molecular Devices
ID3 Multi-mode Plate Reader. % cell viability was calcu-
lated by considering untreated cells as negative control and
geftinib treated cells as positive control.
In contrast to the work reported by Arai et al., present
work reports a reaction that requires only p-TsCl as catalyst
and involves a low-boiling solvent. Arai et al. has reported
benzylation of tryptophan by transesterifcation that requires
addition of nearly same equivalents of p-TsCl as well as
p-TsOH. Addition of p-TsCl to benzyl alcohol forms ben-
zyl p-toluenesulfonate. Since p-toluenesulfonate is a good
leaving group, it is then replaced by carboxylate to form
the benzyl ester of tryptophan. p-TsOH was primarily used
to improve solubility of amino acid in benzyl alcohol in
absence of any solvent, and also to maintain acidic environ-
ment for preventing side-reaction between p-TsCl and amine
function of amino acid (Arai and Muramatsu 1982).
We propose that in presence of p-TsCl catalyst, the ben-
zylation reaction assumes mechanism which is a combina-
tion of transesterifcation and Fischer-Speier esterifcation
Transesterification
O
Fischer-Speier Esterification
O
S Cl
+
HCl
+
OH
S O
O
O
O
O
R1
R1
HCl
OH
OH
NH2
NH2
O
S
O
OH
O
S Cl
O
O
R1
O
O
R1
+
+
HCl
H2O
O
NH2
NH2
Fig. 3 Plausible mechanism of benzyl esterifcation in presence of p-TsCl catalyst
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International Journal of Peptide Research and Therapeutics
(Fig. 3). Benzyl alcohol and p-TsCl form the intermediate
benzyl p-toluenesulfonate. Carboxylate group of amino acid
replaces p-toluenesulfonate to form p-toluenesulfonate salt
of benzyl ester of amino acid as desired product and HCl
as a by-product. The HCl thus formed in situ also behaves
as a catalyst and facilitates the Fischer–Speier esterifcation
reaction in conventionally known way. As water molecule
formed is consumed by p-TsCl to form p-TsOH, azeotropic
distillation is no longer required. When reaction temperature
is allowed to drop to the room temperature, the p-toluenesul-
fonate benzyl ester of amino acid precipitates as a stable salt
on addition of ethyl acetate. Hence, for the aforementioned
reaction p-TsCl catalyst kick starts benzylation of amino
acid via transesterifcation and the HCl formed in situ as a
by-product further catalyses the reaction similar to Fischer-
Speier esterifcation.
For all the synthesized molecules, the base peak in posi-
tive ionisation mode [M+H]+ varied depending upon the
mass of amino acid benzyl ester. In negative ionisation
mode, the precursor m/z [M–H]− was found to be ~ 171.
Anhydrous p-TsOH demonstrates [M–H]− value of 171.01
as its molar mass is 172.2 g/mol. Hence, [M+H]+ val-
ues confrmed O-benzylation of respective amino acids,
while [M–H]− values confrmed their isolation in the form
of stable p-toluenesulfonate salts. Moreover, presence of
singlet peak for methyl protons of p-toluenesulfonate
group is observed in NMR spectra of all the synthesized
compounds at chemical shift values between 2.25 and
2.30 ppm. This observation is consistent with the results
obtained in reported works (Bolchi et al. 2015, 2017).
Hence, the analytical data confrms formation of stable
p-toluenesulfonate salts of O-benzyl amino acids in pres-
ence of p-TsCl catalyst and supports the proposed reaction
mechanism.
Also, the overall pH of reaction mixture is maintained
low not only due to the acidic catalyst p-TsCl but also due
to the by-products like HCl. This ensures absence of any
basic component in the reaction. Maintenance of such acidic
conditions prevent reaction between the chloride catalyst and
amino group, eliminating scope of other by-product forma-
tion (Arai and Muramatsu 1982).
All the synthesized molecules were further subjected to
diferential scanning calorimetry (DSC) studies to confrm
the enantiomeric excess of the synthesized compounds.
Figure 4 gives the representative DSC thermogram for
compound 1a. Compound 1a exhibited a single endother-
mic peak at 130.31 °C, confrming enantiomeric excess of
the analysed compound. DSC thermograms of compounds
1b and 1c (included in Online Resource Figure S10 and
S15) also showed presence of single endothermic peaks at
159.95 °C and 164.91 °C respectively, confrming enan-
tiomeric excess of synthesized compounds.
FTIR spectra recorded for synthesized compounds
showed significant shift in carbonyl- peaks from
νmax ~ 1650 cm−1 to νmax ~ 1750 cm−1, indicating conver-
sion of carboxylic acids to carboxylic esters, respectively.
During mass analysis, p-toluenesulfonates of amino acid
benzyl esters ionized strongly being salts, producing
signals in positive as well as negative ionisation modes.
Fig. 4 DSC thermogram of compound 1a
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International Journal of Peptide Research and Therapeutics
esterifcation of selected amino acids. We propose that in
presence of p-TsCl, benzylation of amino acid assumes a
mechanism which is combination of transesterifcation and
Fischer-Speier esterifcation, and does not require elimina-
tion of in situ formed water by azeotropic distillation. The
p-TsCl catalysed esterifcation resulted into p-toluenesul-
fonates of amino acid benzyl esters with>70% yields. Fur-
thermore, in vitro studies of the synthesized compounds
revealed a possible anticancer activity of 1b (l-aspartic
acid dibenzyl ester p-toluenesulfonate) in A549 (adeno-
carcinoma) cell line, which needs further investigation.
At 25 µM concentrations, all the three compounds were
demonstrated to be safe in 3T3 (fbroblast) cell line, con-
frming their biocompatibility.
Acknowledgements The authors are thankful to the institute Shobha-
ben Pratapbhai Patel School of Pharmacy & Technology Management,
SVKM’s NMIMS University for providing excellent research laboratory
facilities. The authors are also grateful to Mr. Sachin Puri for extending
his help in performing the cytotoxicity assays.
Fig. 5 Results of in vitro cytotoxicity assays of 1a–1c in 3T3 and
A549 cells
In Vitro Assay
Compliance with Ethical Standards
Protected amino acids may be retained as such without
deprotection to form a part of peptide systems designed for
drug delivery (Hegde et al. 2019; Duan et al. 2011). Hence,
we felt the need to confrm biocompatibility of p-toluenesul-
fonates of benzyl-protected amino acids using in vitro cyto-
toxicity assays. Synthesized compounds were investigated
at 25 µM concentrations in A549 (adenocarcinoma) and 3T3
(fbroblast) cell lines using untreated cells as negative con-
trol and geftinib treated cells as positive control. The results
are compiled in Fig. 5.
Conflict of interest Authors declare that they have no confict of inter-
est.
Ethical Approval No experiments involving human participants or ani-
mals were performed for this article.
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